The objective of the proposed research is to understand how the mammalian cell regulates the concentration of the proteins that are externally oriented on the plasma membrane. Toward this objective we have labeled by external labeling methods those proteins that constitute the bulk of plasma membrane proteins in Hepatoma Tissue Culture (HTC) cells. We have analyzed by two-dimensional polyacrylamide gel electrophoretic methods the externally oriented proteins and glycoproteins of the plasma membrane. A complex set of glycoproteins is present on the plasma membrane, but these glycoproteins are not unique to this organelle. By cell fractionation methods and by enzyme probe analyses (with trypsin or neuraminidase) of intact and disrupted cells we can show the existence of a significant intracellular membrane-bound reservoir of these same plasma membrane glycoproteins. The function of this intracellular reservoir of plasma membrane proteins in HTC cells forms a significant portion of the present research proposal. These membrane glycoproteins are being isolated and purified in order to prepare specific antibodies and monoclonal antibodies in mice spleen:MOPC tumor cell hybrids. These antibodies are being used to study the route of biogenesis of these membrane proteins in cells not only growing normally in tissue culture but also after perturbation of the surface concentration of specific membrane glycoproteins by specific antibody or by hormones such as dexamethasone. The exchange of plasma membrane proteins with the proteins of the intracellular pool (via recycling) is also being analyzed. To pursue these studies, we have developed the methodology to insert radioactively-labeled biologically active purified membrane glycoproteins, including those specific receptor functions into the plasma membrane of cells which themselves do not have the specific biological function. With these methods, we have been able to alter the phenotype of the recipient cell to reflect that of the donor cell from which the specific plasma membrane receptor protein was obtained. Our expectation is that the above experiments will shed new and significant insight into the mechanism of biogenesis and function of plasma membrane proteins.